Silylated biomolecule-based hydrogel for culturing cardiomyocytes and stem cells, and use of the hydrogel thereof for treating heart failure

ABSTRACT

The present invention relates to the use of an hydrogel comprising silylated biomolecule for the three-dimensional culture of cardiomyocytes or stem cells which are able to differentiate into cardiomyocytes, and to an aqueous composition comprising i) cardiomyocytes or stem cells which are able to differentiate into cardiomyocytes, and ii) a hydrogel comprising silylated biomolecule, for use for treating heart failure, in particular heart failure following myocardial infarction.

The present invention relates to the use of an hydrogel comprisingsilylated biomolecule for the three-dimensional culture ofcardiomyocytes or stem cells which are able to differentiate intocardiomyocytes, and to an aqueous composition comprising i)cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes, and ii) a hydrogel comprising silylated biomolecule, foruse for treating heart failure, in particular heart failure followingmyocardial infarction.

BACKGROUND OF THE INVENTION

Heart disease, especially myocardial infarction, is a leading cause ofmorbidity and mortality worldwide. Myocardial infarction is an absolutemedical emergency whose incidence remains high with 120,000 cases peryear in France. According to WHO data, on 50 million annual deathsworldwide, ischemic heart disease are the leading cause of death with7.2 million deaths from coronary heart diseases. In France, MI prognosisremains poor, (10 to 12% of total annual mortality in adults). Inaddition, a significant morbidity and socio-economic should be alsoconsidered. Following MI, left ventricular remodeling includes early andprogressive extracellular matrix degradation, infarct zone expansion,scar thinning, and eventually transition to heart failure (Cohn, et al.,2000; Jugdutt, 2003).

Current antiremodeling therapies are clearly limited, because manyventricles continue to enlarge (Bolognese et al., 2007; Savoye et al.,2006) and morbidity and mortality remain high (Verma et al., 2008).Pharmacological treatments currently available can only delay theprogression to end-stage heart failure.

Heart transplantation remains the most effective management of the mostseverely affected patients, but the shortage of donor organs andcomplications associated with this intervention limits this approach.Further, lifelong immune suppression often causes serious complications.

Because the dominant cause of heart failure is loss of myocardium as aresult of infarction and the limited regeneration potential ofcardiomyocytes in mammals, cell therapy may provide a novel therapeuticoption to modify left ventricular remodeling processes and preventpost-infarction heart failure. Thus, in recent years, the possibility ofusing cell transplantation for cardiac repairs has become the focus ofintense research. Multiple cell types have been considered for suchtherapies, including skeletal myoblasts, bone marrow-derivedhaematopoietic stem cells, mesenchymal stem cells, intrinsic cardiacstem cells (CSCs), embryonic stem cells (ESCs) and induced pluripotentstem (iPS) cells.

Conventional administration techniques use intramyocardial injections ofsuspended cells in culture medium. However, this technique is plagued bylimited cell retention and survival. Several studies showed that morethan 80%-90% of grafted cells die within 72 hours after injection intomyocardium (Toma et al., Circulation, 105: 93-98, 2002; Maurel et al.,Transplantation, 80: 660-665, 2005). Further, it was reported thatapproximately 90% of the cells delivered through a needle were lost tothe circulation or leaked out of the injection site (Leor et al.,Circulation, 102: 11156-61, 2000).

In addition, cell-seeded grafts have been proposed for in vitro cardiactissue growth and subsequent in vivo transplantation. These grafts canconsist of embryonic or neonatal cardiomyocytes seeded inthree-dimensional scaffolds; the cardiac myocytes cultured in thesescaffolds can spatially organize and differentiate into myocardium-like3-dimensional tissue. These results suggest that cell therapy and tissueengineering of myocardium have potential for myocardial regeneration orreplacement. However, current approaches to cardiac regeneration faceimportant challenges. Recipient ischemic tissue may be inadequate fordonor cell retention in sufficient quantity to allow for the desiredeffect, because the survival of cells from any source implanted in themyocardium varies between 1% and 10%. Also, nonspecific delivery ofdonor cells to other body sites constitutes an unwanted potential sideeffect.

A particularly useful approach to cardiac regeneration would be a methodthat could employ injection into the injured area in a manner similar tocell injection therapy (rather than surgical implantation ofmyocardium-like volume) and that would provide a suitable growthenvironment for cardiomyocytes.

In recent years, several types of biomaterials, mainly natural proteins,were used in the injectable cardiac tissue engineering, such as fibrin,alginate, matrigel, collagen and chitosan (see for review Wang et al.,J. Cell. Mol. Med., 14(5): 1044-1055, 2010).

However, use of natural materials extracted from biological samples isassociated with a major risk of microbial transmission, essentiallyvirus transmission. Drawbacks associated with natural materials haveprompted the inventors to develop synthetic materials for injectablecardiac tissue engineering.

The inventors had previously developed a silylated hydrogel as a culturematrix for three-dimensional culture of chondrocytes for use forregenerating in vivo cartilaginous tissues (application US2007/0212389).

However, in view of the great difference between cartilaginous tissueand cardiac tissue, respectively hard and soft tissues, and of thephysiological differences between chondrocytes and cardiomyocytes, thelatter needing to retain contractile ability to be functional, it wasnot expected that silylated hydrogel is also usable for culturingfunctional cardiomyocytes.

DESCRIPTION OF THE INVENTION

The inventors have now found that a reticulated hydrogel comprising asilylated biomolecule able to form a pH-dependent self-reticulatinghydrogel can be used for the three-dimensional culture ofcardiomyocytes, or of stem cells which are able to differentiate intocardiomyocytes.

The inventors have also shown that this hydrogel comprising stem cells,in particular mesenchymal stem cells, once injected into the heart, inparticular into left ventricle, has a cardioprotective effect.

The inventors have also shown the tolerability and cytocompatibility ofsilylated biomolecule hydrogel, in particular Si-HPMC hydrogel, withcardiomyocytes, and its ability to maintain a cardiomyocyte phenotypeand to allow physiological cardiomyocyte contractility. Results in vivoalso showed that cells maintained their viability and that hydrogel ofthe invention had no adverse effect on cardiac tissue.

The inventors have also demonstrated that use of hydrogel comprising asilylated biomolecule comprising stem cells able to differentiate intocardiomyocytes, or mesenchymal stem cells, rapidly improves cardiacfunction and preserves long term cardiac function. Further,transplantation of hydrogel comprising a silylated biomoleculecomprising stem cells decreases the infarct expansion, which indicatesthat this combination is capable of preventing negative left ventricularremodeling after myocardial infarction.

Therefore, a first aspect of the present invention relates to the use ofa hydrogel comprising silylated biomolecules, for the three-dimensionalculture of cardiomyocytes or stem cells which are able to differentiateinto cardiomyocytes.

In a preferred embodiment, the cultured cells are cardiomyocytes.

Preferably, the hydrogel comprising silylated biomolecules is able toform a pH-dependent self-reticulating hydrogel at physiological pH (i.e.with a pH value from about 7.2 to about 7.6).

The above use of the invention may be, for instance, an in vitro or exvivo use.

A second aspect of the invention is a method of culturing cardiomyocytesor stem cells which are able to differentiate into cardiomyocytes,comprising the ex vivo mixing of said cells with a hydrogel comprisingsilylated biomolecules at an appropriate pH for forming the hydrogel.Preferably, the pH of the hydrogel comprising silylated biomolecules isa physiological pH (pH from about 7.2 to about 7.6). In a preferredembodiment, the cultured cells are cardiomyocytes.

A third aspect of the invention relates to a method of treating heartfailure, in particular heart failure following myocardial infarction,including administration by injection into myocardium of an aqueouscomposition, preferably at a physiological pH (pH from about 7.2 toabout 7.6), said composition comprising:

i) cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes; and

ii) a hydrogel solution, preferably at physiological pH (pH from about7.2 to about 7.6), comprising silylated biomolecule.

A fourth aspect of the invention concerns an aqueous composition,preferably at a physiological pH (pH from about 7.2 to about 7.6) whichcomprises:

i) cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes; and

ii) a hydrogel solution, preferably at physiological pH (pH from about7.2 to about 7.6), comprising silylated biomolecule;

for use for treating heart failure, in particular heart failurefollowing myocardial infarction.

To carry out the method of treating heart failure and medicalapplication according to the third and fourth aspects of the invention,the aqueous composition will preferably be injectable (i.e. liquid or atleast semi-liquid form, and not totally gelified). The man skilled inthe art can easily determine the appropriate moment when said aqueouscomposition is suitable for being administrated (or used for treating).

Preferably, the aqueous composition is administered (or used fortreating) from a few seconds to 15 minutes after the aqueous solutioncomprising silylated biomolecule is mixed with a buffering solution. ThepH of the final aqueous solution which is administered preferably has aphysiological value (i.e. pH from about 7.2 to about 7.6). Preferably,cardiomyocytes or stem cells are added when the aqueous solution ofpoint (ii) above reaches a pH from 7.2 to 7.6.

Once injected into myocardium, viscosity of the aqueous compositiongradually increases until formation of a hydrogel into whichcardiomyocytes or stem cells which are able to differentiate intocardiomyocytes are present.

The aqueous composition is injected into myocardium, in particular intothe left ventricle, and preferably into the sites along the infarctedarea. The infarcted area can be identified by the surface scar and wallmotion akinesis as described by Takagawa et al. (J. Appl. Physiol.,102(6): 2104-11, 2007, PMID: 17347379).

The injection may be carried out using a system comprising sterilizablesyringe and needle, and connection pieces provided with single-useplungers.

In a preferred embodiment of the method of treating heart failure andmedical application according to the third and fourth aspects of theinvention, stem cells of the aqueous composition are mesenchymal stemcells. The beneficial effects of mesenchymal stem cell grafts are knownto be related in part to their paracrine activity. Mesenchymal stemcells secrete angiogenic, antiapoptotic, and anti-inflammatory cytokinesthat contribute to the recovery of cardiac function and significantlydecrease fibrosis in the heart (Aggarwal S and Pittenger M F., Blood,105: 1815-1822, 2005; Tse W T et al., Transplantation, 75: 389-397,2003; Nagaya N. et al., Circulation, 112: 1128-1135, 2005; and Li L,Zhang Y, Li Y, Yu B, Xu Y, et al., Transpl. Int., 21: 1181-1189, 2008).

A fifth aspect of the invention relates to a kit for obtaining anaqueous composition that is usable in a method of treating heartfailure, in particular heart failure following myocardial infarction,and in the medical application of the invention described above.

The Kit of the fifth aspect of the invention may comprise i)cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes, and ii) a hydrogel solution which comprises silylatedbiomolecule. Preferably, the kit comprises Mesenchymal stem cells.

The kit of the fifth aspect of the invention may also compriseinstructions for the use of said kit in preparing a composition atphysiological pH (pH from about 7.2 to about 7.6) comprising:

i) cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes; and

ii) a hydrogel solution comprising silylated biomolecule;

said composition being intended to be injected into myocardium, inparticular left ventricle.

A sixth aspect of the invention relates to a kit for thethree-dimensional culture of cardiomyocytes or stem cells which are ableto differentiate into cardiomyocytes.

The kit of the sixth aspect of the invention may comprise i)cardiomyocytes or stem cells which are able to differentiate intocardiomyocytes, and ii) a hydrogel solution which comprises silylatedbiomolecule. Preferably, the kit comprises cardiomyocytes or Mesenchymalstem cells, more preferably cardiomyocytes.

The kit of the sixth aspect of the invention may also compriseinstructions for the use of said kit in culturing cardiomyocytes or stemcells which are able to differentiate into cardiomyocytes inthree-dimensional culture

Preferably, the hydrogel solution (ii) of the kits of the fifth andsixth aspects of the invention has a pH higher than 12, and hence is inliquid form (the silylated biomolecule comprised in the hydrogelsolution is stable in aqueous solution at a pH greater than or equal toapproximately 12.4).

Cardiomyocytes or stem cells can be provided in both kits in a culturemedium. The culture media suitable for culturing such cells are wellknown to a person skilled in the art, for instance Dulbecco'S modifiedEagle medium (DMEM), alpha Modified Eagle medium (α-MEM). Alternatively,the cells can be provided as frozen cells.

Both kits may further comprise a buffering solution which may be used toproduce a hydrogel solution ii) with a physiological pH. A suitablebuffering solution may be any solution of pH 4 or below which once mixedwith the hydrogel solution in aqueous solution allow to obtain a finalaqueous solution at physiological pH (i.e. with a pH value from 7.2 to7.6, preferably 7.4) and at physiological osmotic pressure (i.e. about300 mOsm/L). Examples of such a buffering solution are HCl, HEPES andTRIS.

In a preferred embodiment of the first, second and sixth aspects of theinvention, the hydrogel has the following rheological characteristics ata pH value of 7.4 (after 3 weeks of reticulation):

-   -   a compressive modulus at 5% stress from 220 to 15 000 Pa;    -   a storage modulus (G′) from 235 to 10 000 Pa;    -   a loss modulus (G″) from 29 to 1000 Pa;    -   a gel point from 5 to 45 minutes.

More preferably, the hydrogel of the first, second and sixth aspects ofthe invention contains 1 to 3% w/v of si-HPMC (before addition ofcells).

Advantageously, the hydrogel contains 2% w/v of si-HPMC and has thefollowing rheological characteristics at a pH value of 7.4 (after 3weeks of reticulation):

-   -   a compressive modulus at 5% stress from 1600 to 2600 Pa;    -   a storage modulus (G′) from 800 to 2500 Pa;    -   a loss modulus (G″) from 30 to 110 Pa;    -   a gel point from 20 to 30 minutes.

The inventors have found that a hydrogel with the above recitedrheological characteristics is particularly adapted to cardyomyocyteculture since such a hydrogel allows maintenance of cardiomyocytecontractile activity in three-dimensional culture.

In a preferred embodiment of the third, fourth and fifth aspects of theinvention, the hydrogel has the following rheological characteristics ata pH value of 7.4 (after 3 weeks of reticulation):

-   -   a compressive modulus at 5% stress from 220 to 430 Pa;    -   a storage modulus (G′) from 235 to 450 Pa;    -   a loss modulus (G″) from 29 to 60 Pa;    -   a gel point from 23.8 to 30.6 minutes.

When the hydrogel is made of si-HPMC, such rheological characteristicsare obtained when the final aqueous solution (i.e. after addition of thebuffering solution) contains 1.5% w/v of si-HPMC (before addition ofcells).

Preferably, the hydrogel contains 0.8 to 1.5% w/v of si-HPMC (beforeaddition of cells)

The inventors have found that a hydrogel with the above recitedrheological characteristics does not alter MSC viability or activity,and that injection such a hydrogel load with MSCs in the heart directlyafter MI leads to cardiac function and LV remodeling preservation.

The rheological measurements are carried out after 3 weeks ofreticulation, at 25° C., on a rotational rheometer (Rheostress 300,ThermoHaake®, Germany) using a coni-cylindrical geometry with a diameterof 60 mm and a cone angle of 1°. A multiwave procedure with 3frequencies 1, 3.2 and 10 Htz is used, and the imposed stress is 1 Pa.Oscillation tests measuring storage modulus (G′) and loss modulus (G″)are performed to study the self-setting process and gel point. The gelpoints are given as the time taken for the liquid (G″>G′) to turn into asolid (G′>G″). They are determined according to a derived percolationtheory as disclosed by Fatimi et al. (Acta Biomater, 5: 3423-3432).Compressive modulus of scaffold is measured using a TA HD-Plus (StableMicro Systems). The compressive modulus is calculated on the basis ofstrain change from 0 to 5%. Shear strain measurements are performed witha Haake mars. Frequencies are applied at a fixed total shear stress (1Pa) and 0.21N. Oscillation tests are performed to measure G′ and G″after 3 weeks of gelation.

Preferably, the hydrogel and hydrogen solution comprising silylatedbiomolecules used in the different aspects of the invention are able toform a self-reticulating hydrogel at a pH between 7 and 12.

As used herein, the terms <<hydrogel>> and <<hydrogel solution>> whichare used indifferently mean a network of polymer chains that arewater-insoluble, in which water is the dispersion medium.

As used herein, the term “silylated biomolecules” means any organic orsynthetic molecules onto which are grafted a silyl function, preferablyan alkoxysylane. Silylation allows the formation of covalent bondsbetween the biomolecules constituting the hydrogel as a function of pH.The silylated biomolecules are thus able to form a pH-dependentself-reticulating hydrogel.

The term “organic molecule” is intended to mean any molecule that isproduced by a living organism or that is a derivative thereof, includinglarge polymeric molecules such as proteins, polysaccharides, and nucleicacids as well as small molecules such as primary metabolites, secondarymetabolites, and natural products.

The term “synthetic molecule” is intended to mean any molecule that isproduced by chemical methods, such as proteins, polysaccharides, nucleicacids and a mix thereof.

As examples of biomolecules, mention may be made of:

-   -   lipid derivatives such as phospholipids, glycolipids and        sterols,    -   chemical messengers such as hormones and neurotransmitters,    -   vitamins,    -   sugar derivatives such as carbohydrate, disaccharide,        oligosaccharides, polysaccharides (including cellulose),    -   amino acid derivatives such as amino acids (natural and/or        non-standard), peptides, oligopeptides, polypeptides, proteins        (said peptides, oligopeptides, polypeptides and proteins        containing natural and/or non-standard aminoacid),    -   nucleotides derivatives such as nucleotides, and biological        polymers such as deoxyribonucleic acid (DNA), ribonucleic acid        (RNA),    -   biopolymers such as lignin, proteins, DNA, RNA,        oligosaccharides, polysaccharides.

Preferably, the biomolecule is a polysaccharide, a protein, or apeptide.

As used herein, the term “polysaccharide” means a polymer made up ofmany monosaccharides joined together by glycosidic bonds. Natural andsynthetic polysaccharides are included. Examples of polysaccharide arecellulose and derives thereof, for instance hydroxypropylmethylcellulose(HPMC), hydroxyethylcellulose (HEC), and carboxymethylcellulose (CMC),pectin, chitosan and hyaluronic acid.

As used herein, the term “protein” means a polymer made of amino acidsarranged in a linear chain and joined together by peptide bonds betweenthe carboxyl and amino groups of adjacent amino acid residues.Glycoprotein as well as proteins containing natural and/or non-standardaminoacid are included. Albumin, laminin, gelatin, fibronectin,vitronectin and collagen are examples of protein.

The hydrogel may contain either only one kind of silylated biomolecule(i.e. all the biomolecules forming the polymer are the same), orsilylated biomolecules of different nature, preferably two differentsilylated biomolecules, more preferably one silylated polysaccharide andone silylated protein or peptide.

In a preferred embodiment, the hydrogel contains a silylated HPMC, asilylated HEC or a silylated CMC polymer.

In a particularly preferred embodiment, the hydrogel contains onlysilylated HPMC.

In another preferred embodiment, the hydrogel is formed with:

-   -   silylated collagen and silylated HPMC (leading to a hydrogel        containing HPMC and collagen),    -   silylated hyaluronic acid and silylated HPMC (leading to a        hydrogel containing HPMC and hyaluronic acid),    -   silylated tetrapeptide Arg-Gly-Asp-Ser (hereafter abbreviated        “RGDS”) and silylated HPMC (leading to a hydrogel containing        HPMC and RGDS), or    -   silylated pectin and silylated hyaluronic acid (leading to a        hydrogel containing pectin and hyaluronic acid).

The silylated biomolecules used to carry out the invention arepreferably stable in aqueous solution at a pH greater than or equal toapproximately 12.4.

Thus, the aqueous solution comprising silylated biomolecules of theinvention are preferably liquid at a pH of about 12.4 or above.

As used herein, the expression “aqueous solution” means a solutionwherein water is the major solvent.

Acidification of the solution causes a gradual increase in viscosity,due to the condensation of the silylated biomolecules via the formationof —Si—O—Si— covalent bond, and the formation of hydrogel. Becausehighly basic pH is detrimental for cells, even when cells are exposed toit for a short period, the silylated biomolecules is contacted with anacid in an aqueous solution to obtain a final aqueous solution atphysiological pH (i.e. with a pH value from 7.2 to 7.6, preferably 7.4),before cardiomyocytes or stem cells are added. The hydrogels and theaqueous solution comprising silylated biomolecules of the invention arepreferably capable of gelation at physiological pH.

Addition of cells must occur before the total gelation of the finalsolution to hydrogel, preferably from a few seconds to 15 minutes afterhaving contacted the silylated biomolecules with an acid and havingobtained a final aqueous solution of pH from 7.2 to 7.6.

Processes for preparing silylated biomolecules and hydrogels accordingto the invention are well known by one skilled in the art. For instance,processes for the preparation of silylated HPMC and silylated HEC andfor the preparation of hydrogel therefrom are described in USapplication 2007/0212389.

Methods for preparing silylated biomolecules are also described inapplication PCT/EP2011/050981. PCT/EP2011/050981 also disclosed thepreparation of hydrogel comprising two different kinds of silylatedbiomolecules, in particular a silylated polysaccharide and a silylatedprotein or peptid.

Depending on whether the biomolecule used as starting material iscarrying an amine or an alcohol function, or a carboxylic acid function,the two processes described below (and disclosed in applicationPCT/EP2011/050981) may be used to prepare silylated biomolecules.

For preparing a silylated biomolecule using a biomolecule carrying analcohol or amine function, preferably chosen from a peptide, anoligopeptide, a protein, a deoxyribonucleic acid, a ribonucleic acid,pectin, chitosan, hyaluronic acid, a glycolipid as a starting material,the process (hereafter called “process 1”) comprises a step of reactingsaid biomolecule with a silylation agent having the following formula(II) or (IIbis):

wherein:

-   -   m is an integer ranging from 1 to 6,    -   p and q are independently 0 or 1, and    -   R₁, R₂ and R₃ each independently represent a C₁-C₆ alkyl group.

Silylated biomolecules of formula (I):

wherein:

-   -   A is a biomolecule chosen from a peptide, an oligopeptide, a        protein, a deoxyribonucleic acid, a ribonucleic acid, pectin,        chitosan, hyaluronic acid, a glycolipid, and a polysaccharide    -   m is an integer ranging from 1 to 6,    -   p and q are independently 0 or 1,    -   X is a —NHCONH— or a —OCONH— moiety, and    -   R₁, R₂ and R₃ each independently represent a C₁-C₆ alkyl group.        are obtained by process 1.

During the process, the amine or the alcohol function of the biomoleculereacts with the isocyanate function of the silylation agent of formula(II), leading to the formation of an urea bond (—NHCONH—) (if thebiomolecule is carrying an amine function) or a carbamate bond(—OCONH—)(if the biomolecule is carrying an alcohol function) accordingto the following scheme:

In one embodiment of process 1, the biomolecule is carrying an alcoholfunction and is preferably chosen from a deoxyribonucleic acid, aribonucleic acid, pectin, chitosan, hyaluronic acid, a glycolipid andoptionally from a peptide, an oligopeptide, a protein, when saidpeptide, oligopeptide, or protein comprise a moiety (an amino acid forexample) carrying an alcohol function, for example the RGDS.

In one other embodiment of process 1, the biomolecule is carrying anamine function and is preferably chosen from a peptide, an oligopeptide,a protein, a deoxyribonucleic acid, a ribonucleic acid and chitosan. Thebiomolecule used in the process can also carry both an alcohol functionand an amine function, for example when the biomolecule is chitosan.

In a preferred embodiment of process 1, the silylation agent used in theprocess is 3-isocyanatopropyltriethoxysilane.

Preferably, when the biomolecule is carrying amine functions, part ofsaid amine functions are not protonated in the reaction medium. The lonepair of the amine has indeed to be available to attack the isocyanatefunction.

The temperature of the reaction of process 1 is not critical and mayvary in wide range. The reaction is generally carried out at atemperature from −15° C. to 40° C., preferably 0° C. to 30° C., morepreferably from 15° C. to 25° C., which is advantageous as nodenaturation of biomolecule occurs. Preferably, process 1 is carried outunder inert atmosphere, for example under argon or nitrogen.

The reaction time is usually lasts from one hour to one week, preferablyfrom twelve hours to five days, more preferably from one to three days.

Process 1 is generally carried out in a solvent. There is no particularrestriction on the nature of the solvent to be used, provided that ithas no adverse effect on the reaction or on the reagents involved.Organic solvents or mixture of organic solvent with an aqueous solution,typically water, are preferred. Examples of suitable organic solventsinclude acetonitrile, acetone, dimethylformamide and dimethylsulfoxide.

In one embodiment, process 1 is carried out in an anhydrous solvent,such as anhydrous acetonitrile, anhydrous acetone, anhydrousdimethylformamide or anhydrous dimethylsulfoxide, and in the presence ofa base, preferably an organic base, usually an organic base containing anitrogen atom which can be protonated, for example triethylamine,pyridine or trimethylamine.

In one other embodiment, process 1 is carried out in a mixturecomprising an aqueous solution and a solvent miscible in water, such asacetonitrile, acetone, dimethylformamide and dimethylsulfoxide. Themixture is preferably a mixture of water and of dimethylsulfoxide. Nobase is required for this embodiment.

For preparing a silylated biomolecule using as starting material abiomolecule carrying a carboxylic acid or a carboxylate function, aprocess is described therein (hereafter called “process 2”). Thisprocess for the preparation of a silylated biomolecule of formula (I) asdefined above, comprising the steps consisting of:

a) reacting a biomolecule carrying a carboxylic acid or a carboxylatefunction, preferably chosen from a peptide, an oligopeptide, a protein,pectin, hyaluronic acid, with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) orwith 1,1′-carbonyldiimidazole (CDI), then

b) adding to the reaction medium obtained in step a) a silylation agenthaving the following formula (III):

wherein,

-   -   n is an integer ranging from 1 to 6    -   p and q are independently 0 or 1, and    -   R₁, R₂ and R₃ each independently represent a C₁-C₆ alkyl group.

Silylated biomolecules of formula (I), wherein X is a —CONH— moiety, areobtained by process 2. During process 2, the carboxylic function of thebiomolecule is activated with EDC.HCl in step a) and then reacts withthe amine function of the silylation agent of formula (III), leading tothe formation of an amide bond (—CONH—) according to the followingscheme:

Preferred biomolecule used as starting material in step a) of process 2are a peptide, an oligopeptide, a protein, pectin, and hyaluronic acid.

Step a) of process 2 can be carried out in the presence of a catalyst,such as N-hydroxysuccinimide.

The silylation agent used in step b) of process 2 is preferably(3-aminopropyl)triethoxysilane.

When EDC.HCl is used, steps a) and b) of process 2 are generally carriedout in an aqueous solution, the pH of which is preferably from 4 to 6,most preferably from 4.7 to 5.3, preferably in water. When CDI is used,steps a) and b) of process 2 are generally carried out indichloromethane or acetonitrile.

Steps a) and b) of process 2 are generally carried out at a temperaturefrom −15° C. to 40° C., preferably 0° C. to 30° C., more preferably from15° C. to 25° C., which is advantageous as no denaturation ofbiomolecule occurs.

Step a) of process 2 usually lasts from 4 h to 24 h, preferably from 12h to 18 h, and step b) of process 2 usually lasts from 4 h to 24 h,preferably from 12 h to 18 h.

Both processes 1 and 2 lead to the formation of a strong covalent bondbetween the silylation agent and the biomolecule.

The weight concentration of the biomolecule used as starting material inthe solvent in processes 1 and 2 is generally from 0.01 to 30%,preferably from 0.1 to 20%, more preferably from 0.5 to 15%.

Advantageously, processes 1 and 2 are carried out without any metalcatalyst, more particularly tin based catalyst.

When process 2 wherein EDC. HCl is used is carried out, the reactionmedium is generally homogeneous. When process 1 or process 2 wherein CDIis used are carried out, the reaction medium is generally heterogeneous.A suspension of the biomolecule in the solvent is generally observed,which can be isolated easily from the reaction mixture, for example bysedimentation or centrifugation.

The cardiomyocytes and the stem cells which are able to differentiateinto cardiomyocytes used in the different aspects of the invention maybe, for example, human, non human primate, rat, dog, mouse, or catcells, more preferably human cells.

The stem cells may be, for example, embryonic stem cells, or adult stemcells such as skeletal myoblasts (stem cells from muscle),bone-marrow-derived stem cells (in particular mesenchymal stem cells),adipose-derived mesemchymal stem cells, cardiac stem cells (disclosed byBeltrami et al., Cell, 114: 763-776, 2003).

In a preferred embodiment, stem cells are mesenchymal stem cells, morepreferably bone-marrow-derived mesenchymal stem cells.

Methods for isolating embryonic and adult stem cells are well known bythe person skilled in the art, and are for example disclosed inPittenger et al. (Circ Res., 95(1): 9-20, 2004, PMID: 15242981), and inBlin et al. (J. Clin. Invest., 1; 120(4):1125-39, 2010 doi:10.1172/JCI40120. Epub 2010 Mar. 24. PMID 20335662).

The stem cells may also be induced pluripotent stem cells (hereafterabbreviated “iPS”), that is population of cells with characteristicsreminiscent of embryonic stem cells which is generated from somatictissues through nuclear reprogramming via the ectopic expression ofgenes related to pluripotency. Processes for generating iPS cells arefor instance described by Takahashi et al. (Cell, 131: 861-872, 2007),Yu et al. (Science, 318: 1917-1920, 2007) and Okita et al. (Nature, 448:313-317, 2007).

In embodiments of the different aspects of the invention wherecardiomyocytes are used, said cardiomyocytes may be derived from stemcells or iPS cells.

Further, in embodiments of the different aspects of the invention wherestem cells able to differentiate into cardiomyocytes are used, thehydrogel may comprise agents which allow induction of cardiomyocytedifferentiation, so that the final cells obtained are cardiomyocytes.Examples of agents used in protocols of cardiac differentiation of stemcells are disclosed by Blin, et al. (Curr. Stem Cell Res. Ther.,5(3):215-26, 2010). Preferably, where stem cells able to differentiateinto cardiomyocytes are mesemchymal stem cells, in particularadipose-derived mesemchymal stem cells, the hydrogel does compriseagents which allow induction of cardiomyocyte differentiation.

Buffering solutions which may be used to produce a final aqueoussolution with a physiological pH are well known to one of ordinary skillin the art. Examples of such a buffering solution are4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, HCl).

The present invention will be further illustrated by the additionaldescription and drawings which follow, which refer to examplesillustrating the characterization of the properties of a hydrogelaccording to the invention comprising cardiomyocytes or stem cells, andits use to preserve cardiac function and left ventricular remodeling inacute stage following myocardial infarction. It should be understoodhowever that these examples are given only by way of illustration of theinvention and do not constitute in anyway a limitation thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates viability of cardiomyocytes cultured with or withoutSi-HPMC.

Cardiomyocytes were cultured in 2D with or without Si-HPMC (control) orin the presence of actinomycin-D (5 μg/ml) during the indicated times.Viability was assessed by MTS activity. Results are expressed asrelative MTS activity compared with the respective control. *P<0.001 ascompared to control conditions.

FIG. 2 illustrates a real-time RT-PCR analysis of cardiomyocytephenotype in culture in 2D with the Si-HPMC hydrogel. mRNA was harvestedfrom cardiomyocytes after 1, 2 and 3 days of culture with or withoutSi-HPMC (control). Real-time RT-PCR was performed by using nkx2.5,gata4, cardiac sarcomeric α-actin and connexin43 and corrected by HPRTgene expression levels. *P<0.001.

FIG. 3 illustrates the quantification of cardiomyocyte contractility in2D culture. The contractility was quantified manually by counting thecardiomyocytes beats for one minute. This frequency was measured after24 and 48 hours of culture in 2D of cardiomyocytes with or without(control) Si-HPMC. Results are expressed as beat number/minute. *P<0.01.

FIG. 4 illustrates the three-dimensional cellular viability of MSCs inSi-HPMC hydrogel. MSCs were cultured in 3D into Si-HPMC hydrogel duringthe indicated times. Cells were stained with calcein-AM and EthD-1,which label living cells in green and dead cells in red, respectively.MSC viability was assessed by the intensity of green fluorescence, as aconsequence of incorporation of the calcein fluorescent probe into cellcytoplasm. Percentages of living and dead MSCs cultured in 3D withinhydrogel over 7 days (p=NS as compared between groups).

FIG. 5 illustrates measurements of VEGF protein concentrations by ELISAassay. VEGF concentrations in (A) control supernatants of MSCs culturedwithout hydrogel (p<0.001 for all comparisons) and in (B) supernatantsof MSCs cultured in 3D within hydrogel (p<0.001, for all comparisons).VEGF concentrations were expressed as pg·ml⁻¹ for 10⁴ cells.

FIG. 6 illustrates the evaluation of cardiac function byechocardiography in rats after myocardial infarction (MI). Measurementswere performed at baseline before MI and 1, 7, 28 and 56 days after MI.(A) LV end-diastolic diameter (LVEDD), (B) LV end-systolic diameter(LVESD). (C) The fraction shorting (FS) and (D) Ejection fraction (EF).^(¥)p<0.05 compared to day 1 post-infarction in the same group, *p<0.001compared to the PBS group at the same time-point, ^($)p<0.05 compared tothe Hydrogel group at the same time-point and ⁺p<0.05 compared to theMSCs at the same time-point.

FIG. 7 illustrates the effects on Myocardial Infarction and fibrosis ofinjection of PBS (control group), Hydrogel, MSC, or MSC+hydrogel intomyocardium of rats suffering from myocardial infarction. (A)Representative histologic sections of Masson trichrome staining forinfarct size measurement (collagen-rich areas in blue and healthymyocardium in red; original magnifications: ×40). (B) Circumferentialinfarct size (MI size) to total LV tissue and (C) percentage of fibrosisto LV tissue. For (B) and (C): *p<0.05 and **p<0.001 compared to the PBSgroup and ^($)p<0.05 and ^($$)p<0.001 compared to Hydrogel group.

FIG. 8 illustrates the effects on scar thickness and infarct expansionof injection of PBS (control group), Hydrogel, MSC, or MSC+hydrogel intomyocardium of rats suffering from myocardial infarction. (A)Representative photomicrographs of Masson trichrome staining of the scararea (collagen-rich areas in blue and healthy myocardium in red;Original magnifications: ×100). (B) Relative scar thickness (averagescar thickness/average wall thickness). (*p<0.05 and **p<0.001, two-wayANOVA). (C) Infarct expansion index ([LV cavity area/whole LVarea]/relative scar thickness). For (B) and (C): *p<0.05 and **p<0.001.

EXAMPLES Example 1: Preparation of Hydrogel

Materials

-   -   HPMC E4M™ (Colorcon-Dow Chemical, France)    -   Glycidoxypropyltriméthoxysilane (GPTMS) (Acros, Belgium)    -   HEPES and HCl (Sigma-Aldrich, St Louis, the USA)    -   NaOH and NaCl (International VWR, Fontenay-under-Wood, France)

Synthesis of Si-HPMC Hydrogel

As previously described (Bourges et al., Adv. Colloid Interface Sci.,99: 215-228, 2002), the synthesis of Si-HPMC was performed by grafting14, 24% of 3-GPTMS on E4M® in heterogeneous medium. Aqueous solution ofSi-HPMC was prepared at 3% w/w concentration. The powder was dissolvedin sodium hydroxide solution (0.2M NaOH) at 25° C. for 48 h. Si-HPMCsolution was then dialyzed in a dialysis bag against 3.81 of NaOHsolution (0.09M) for 12 h and with 4 L of NaOH solution (0.09M) for 2 h.The solution was then sterilized by steam (121° C., 30 mn). To allow theformation of a reticulated hydrogel, 1 volume of the solution wasfinally mixed with 1 volume of a 0.13 M HEPES buffer.

Rheological Measurements

Dynamic rheological measurements were performed on a Haake Rheometer(rheostress 300) using a coni-cylindrical geometry with a diameter of 60mm and a cone angle of 1°. We used a multiwave procedure with 3frequencies 1, 3.2 and 10 Hz, and the imposed stress was 1 Pa.Oscillation tests measuring storage modulus (G′) and loss modulus (G″)were performed to study the self-setting process and gel point.Compressive modulus of scaffold was measured using a TA HD-Plus (StableMicro Systems). Six specimens were tested after three weeks ofreticulation and the compressive modulus was calculated on the basis ofstrain change from 0 to 5%. Shear strain measurements were performedwith a Haake mars. Frequencies were applied at a fixed total shearstress (1 Pa) and 0.21N. Oscillation tests were performed to measure G′and G″ after 3 weeks of gelation. Nine specimens were tested.

Results:

Rheological properties of Si-HPMC hydrogel mixed with one volume of a0.13M buffer (1v1) were measured. The compressive modulus at 5% stressand the storage modulus (G′) and loss modulus (G″) of Si-HPMC wereperformed after three weeks of reticulation.

The final product (Si-HPMC) consisted of a reticulated hydrogel after27.2±3.4 min with a pH value of 7.4. Dynamic rheological measurementswere performed to characterize this hydrogel. Shear strain measurementswere performed to determine de storage modulus (G′), which characterizedthe hard component and the loss modulus (G″), which characterized theliquid component. The compressive modulus reflects the capacity of amaterial to resist to strengths. When the limit of the compressivestrength is reached, the hydrogel is destroyed. In the case of ourSi-HPMC hydrogel, compressive modulus was about 328.56±96.97 Pa. Afterthree weeks of reticulation and a finished self-setting process, weobserved a value of 343.17±106.5 Pa for the storage modulus (G′) and avalue of 44.48±15.43 the loss modulus (G″).

Example 2: Preparation and Cell Culture

Materials

-   -   Dulbecco'S modified Eagle medium (DMEM), alpha Modified Eagle        medium (α-MEM)    -   Hank's Balanced sodium salt (HBSS), horse serum,        Penicillin/streptomycine, L-glutamine, collagenase II (284.00        unit/mg), Trypsine/EDTA (Invitrogen corporation, Paisley, the        U.K.)    -   pancreatin (0.1 mg/ml), laminin (Sigma-Aldrich, St-Louis, USA)    -   Fetal Calf Serum (FCS) (Hyclone Perbio, Thermo Fisher        scientific)    -   Animals: neonatal C57BI/6j mice and Lewis female rats (Janvier,        France)

Isolation and culture Cardiomyocytes:

Primary cardiomyocytes were isolated from 1 or 2-day-old neonatalC57B1/6j mice hearts. Briefly, neonatal mice were sacrificed and heartswere rapidly removed and placed into dishes on ice. After atria andgreat vessels were removed, hearts were minced and digested repeatedly(10 min×8) in HBSS solution supplemented with collagenase 11 (284.00unit/mg) and pancreatin (0.1 mg/ml) at 37° C. and 5% CO2. Aftercentrifugation, cells were resuspended in culture media (DMEM with 10%horse serum, 5% SVF, 1% penicilline/streptomycine).

For two dimensional culture with the Si-HPMC hydrogel, cardiomyocyteswere plated in 24-well plates (coated with laminin 10 μg/ml) at thedensity of 55 000 cells/cm² and maintained at 37° C. in a humidifiedatmosphere and 5% CO2. After 48 hr, culture medium was removed and 500μl of Si-HPMC were added in each well. Samples were incubated at 37° C.for 1 h before adding 500 μl of culture medium. For 3D-culture ofsuspended cardiomyocytes in Si-HPMC hydrogel, 10 μl of culture mediumcontaining 9×10⁶ cardiomyocytes were mixed with 1 ml of Si-HPMC. 500 μlof cells/Si-HPMC mixture were seeded in 12-well plates and incubated at37° C. and 5% CO2. After 1 hr incubation, 1 ml of culture medium wasadded in each well and plates were incubated. For cardiomyocyte3D-culture in a micro-drop of Si-HPMC hydrogel, 5 μl of culture mediumcontaining 2×10⁴ cardiomyocytes were directly injected in a micro-dropof Si-HPMC after 2 hours of polymerization.

Isolation and Culture of MSC:

Bone marrow (BM) was obtained from Lewis female rats weighing 180-200 g.BM from femurs cavity was flushed with α-MEM medium containing 10% FBSand 1% penicillin/streptomycin, and the cell suspension was centrifuged(1200 rpm, 7 min). Cells were then plated in culture flasks (200 000cells/cm2). Non adherent cells were removed after 72 hours, and MSCswere recovered by their capacity to strongly adhere to plastic culturedishes. MSCs were then routinely cultured and were used for experimentsafter the third passage.

Example 3: Study of the Cytotoxicity of Hydrogel

Materials

-   -   Plate culture 24 wells Corning-Costar (Corning BV,        Schiphol-Rijk, The Netherlands).    -   Actinomycin D and Dyméthylsulfoxyde (DMSO) (Sigma-Aldrich)    -   Methyl Tetrazolium Salt (MTS) (Titer Concealment 96 MTS, Promega        corporation, Madison, Wis.).    -   Buffered salt phosphates (PBS, Invitrogen corporation).

A. Cardiomyocyte Viability:

Cardiomyocyte viability in 2D culture was measured using an MTS assay aspreviously described (Relic et al., 2001; Magne et al., 2003). As acontrol, cells were also cultured in the absence of Si-HPMC or in thepresence of actinomycin-D (5 mg/ml), an inhibitor of RNA polymerase(Kimura et al., 2002) used as a potent inducer of cell death. After 24and 48 hours, hydrogels and culture media were removed and MTS solutionwas added in each well for 1-3 h according to the manufacturer'sinstructions. Finally, colorimetric measurement was performed on aspectrophotometer at an optical density of 490 nm. Results wereexpressed as relative MTS activity compared to control condition (cellscultured in the absence of Si-HPMC).

Results:

Cardiomyocyte viability was evaluated using MTS activity at 24 and 48hours of 2D culture in presence of Si-HPMC hydrogel. No significantdifference was observed between control cultures and the culturescarried out in contact with hydrogel (see FIG. 1). On the other hand,the actinomycin-D, inhibitor of the transcription, used here ascytotoxicity positive control induced a significant reduction in MTSactivity of cardiomyocytes after 24 hours of culture. In the presence ofactinomycin D MTS activity decreased by nearly 55% after a 24 htreatment and by 90% after a 48 h treatment. Therefore, the Si-HPMChydrogel maintains cardiomyocyte viability.

B. Cardiomyocyte Phenotype

-   -   Transcripts analyses:

Materials:

-   -   RNeasy Mini Kit (Qiagen S.A., France)    -   High-capacity cDNA Archive kit (Applied Biosystems, life        technologies corporation, USA)    -   Taqman gene expression (Applied Biosystems, life technologies        corporation, USA)

In order to analyze cardiomyocyte phenotype, expression of mRNA codingfor cardiomyocyte markers was quantified by RT-PCR. RT-PCR analysis oftranscripts was performed on cardiomyocytes in 2D culture in the absenceor presence of Si-HPMC.

Total RNA extraction and DNAse treatment Total RNAs from each cardiacsample were isolated and DNase-treated with the RNeasy Fibrous TissueMini Kit following manufacturer's instructions.

Reverse transcription: First-stand cDNA was synthesized from 200 ng oftotal RNAs using the High-capacity cDNA Archive kit.

Reaction of polymerase in chain (PCR) On-line PCR was performed usingthe following primers: nkx2.5 (nkx2.5, Mm00657783_m1), gata4 (gata4,Mm00484689_m1), actin alpha cardiac muscle 1 (actc1, Mm01333821_m1), gapjunction protein alpha 1 (gja1, Mm00439105_m1). Fluorescence signalswere normalized to the hypoxanthine guanine phosphoribosyl transferase 1(hprt1, Mm03024075_m1), used as reference gene. Data were averaged andthen used for the 2^(−ΔCT) calculation. 2^(−ΔCT) corresponded to theratio of each gene expression versus hprt.

Results:

The ability of Si-HPMC to maintain cardiomyocyte phenotype after 1 day,2 and 5 days of 2D culture was evaluated by relative quantification ofcardiogenic marker (nkx2.5, gata-4, cardiac sarcomeric α-actin andconnexin 43) mRNAs, using TaqMan real-time PCR (see FIG. 2). Expressionlevels of these cardiomyocyte markers were maintained during the 5 daysof culture in presence or absence d'HPMC. Importantly, the presence ofthe Si-HPMC hydrogel did not alter expression levels of these genes incardiomyocytes.

-   -   Immunostaining:

Materials:

-   -   Formaldehyde solution 37% (Sigma-Aldrich)    -   Triton X-100 (Sigma-Aldrich)    -   Bovine serum albumin (Sigma-Aldrich)        -   Polyclonal antibodies: anti-nkx2.5 and anti-gata4 (Santa            Cruz

Biotechnology, USA).

-   -   Monoclonal anti-connexin 43 (Millipore)    -   Secondary antibodies Alexa fluor (Molecular Probes, Leiden, The        Netherlands)    -   Vectaschield® medium with DAPI nuclear (vector laboratories, US.        Headquaters).

Cardiomyocytes were fixed in 4% formaldehyde for 30 min at roomtemperature and permeabilized with 0.2% Triton X-100, bovine serumalbumin, and phosphate-buffered saline (BSA-PBS). then, cells wereincubated for 1 h at room temperature with primary antibodies:polyclonal anti-nkx2.5 (1:500), polyclonal anti-gata4 (1:500),monoclonal anti-connexin 43 (1:100) and monoclonal anti-sarcomeric alphaactin (1:1000). Cells were washed and incubated for 45 min at roomtemperature with fluorescence-conjugated secondary antibodies at a1:1000 dilution: Alexa fluor 568 mouse anti goat IgG, Alexa Fluor 488goat anti-mouse IgG and Alexa fluor 594 goat anti-mouse IgG. Cells werewashed carefully with PBS and the samples were mounted withVectaschield® medium with DAPI nuclear. Cardiomyocytes were observedusing fluorescence microscopy, and pictures were taken using a ZeissAxioskop2 with equal exposure times. The extent of fluorescence wasmeasured by using MetaMorpho microscope image analysis software (version6.3).

Results:

The expression and localization of proteins nkx2.5, gata4, cardiacsarcomeric α-actin and connexin 43 were observed by immunofluorescencestaining after 48 hours of culture (data not shown). The expression ofboth transcription factors nkx2.5 and gata-4 were maintained in thenuclei of cardiomyocytes cultured with Si-HPMC, as well as membraneexpression of connexin 43. Staining for sarcomeric α-actin revealedtypical sarcomeric striations in cardiomyocytes cultured in presence orabsence of Si-HPMC. These results suggest Si-HPMC hydrogel maintainedcardiomyocyte phenotype.

C. Cardiomyocyte Contractility

After 24 and 48 hours of culture in the presence or absence Si-HPMC,cardiomyocytes were observed by videoscopy using a Nikon eclipse TE200Emicroscope. Spontaneous contractions were quantified over one minute.Functional activity of 3D cultured cardiomyocytes was visualized usingvideoscopy after 48 hours of culture.

Results:

Cardiomyocyte contractility was qualitatively and quantitativelycharacterized by image analysis of the contraction videos. After 24hours of 2D culture, cardiomyocytes began to display spontaneouscontractions and after 48 hours their contractile activity wassynchronous. Contraction rate was almost similar when cardiomyocyteswere cultured in the absence or presence of the Si-HPMC hydrogel (seeFIG. 3) (140 beats/min at 24 hrs and 80 beats/min at 48 hrs). The seededcardiomyocytes suspended in Si-HPMC hydrogel showed a round morphologysince these cells could not adhere to matrix. In addition, cells hadvery few intercellular contacts which prevented evaluation ofelectromechanical coupling. However after 48 hours of culture, severalcardiomyocytes showed spontaneous contractile activity, cells hadmigrated and created contacts with neighboring cells favoringcontraction. To promote electromechanical coupling between cells,cardiomyocytes were seeded into micro-droplets in the hydrogel. After 48hours of culture, clusters of cells with synchronous contractility wereobserved. These results suggest that Si-HPMC hydrogel allows maintenanceof cardiomyocyte contractile activity in 2D and 3D culture.

Example 4: Injection of Si-HPMC Hydrogel with MSC in Myocardium

Materials and Methods

Isolation and Culture of BM-MSC

Bone marrow (BM) was obtained from Lewis female rats weighing 180-200 g(Janvier France, http://www.janvier-europe.com). BM from femur cavitywas flushed with α-MEM medium (Invitrogen corporation, Paisley, the U.K)containing 10% FCS (Hyclone Perbio, Thermo Fisher scientific), 1%L-Glutamin, 1% penicillin/streptomycin (Invitrogen) and 2 ng/ml of humanFGF2 (AbCys P100-18B). The cell suspension was centrifuged (1200 rpm, 7min). Cells were then plated in culture flasks (200 000 cells/cm2). Nonadherent cells were removed after 72 hours, and mesenchymal stem cells(MSCs) were recovered by their capacity to strongly adhere to plasticculture dishes. MSCs were then routinely cultured and were used forexperiments after verification of their phenotype by flow cytometricanalysis for surface markers (CD29, CD45, CD90 and Sca1) at passage 3.

Silanized Hydroxypropyl Methylcellulose-Based Hydrogel Preparation

Synthesis of Si-HPMC Hydrogel

Hydroxypropyl methylcellulose (HPMC) E4M® was purchased fromColorcon-Down chemical (Bougival, France). The synthesis of Si-HPMC wasperformed by grafting 0.5% of silicium in weight on HPMC (E4M®)heterogeneous medium, as previously described by Boor P J, and Ferrans VJ. (Am. J. Pathol., 121: 39-54, 1985) (Si-HPMC powder 3%) wassolubilized in 0.2M NaOH under constant stirring for 48 h. The solutionwas dialyzed against 0.09 M NaOH using 6-8 kDa dialysis tubes(SpectraPor 1, Fisher Scientific, France). The resulting viscoussolution (pH 12.6) and a 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid buffer (HEPES, pH 3.6; Sigma-Aldrich, St Louis, the USA) wereseparately steam sterilized by steam (121° C., 30 min) and then mixedusing luer-lock syringes at a volume ratio of 1/1 as previouslydescribed by Bourges et al. (Adv. Colloid Interface Sci., 99: 215-228,2002). Final product consists in hydrogel (pH=7.4) containing Si-HPMCconcentration of 1.5%.

Rheological Measurements

Reticulation of 1 ml Si-HPMC was induced in 12-well plates. Dynamicrheological measurements were performed on a rotational rheometer(Rheostress 300, ThermoHaake®, Germany) using a coni-cylindricalgeometry with a diameter of 60 mm and a cone angle of 1°. We used amultiwave procedure with 3 frequencies 1, 3.2 and 10 Htz, and theimposed stress was 1 Pa. Oscillation tests measuring storage modulus(G′) and loss modulus (G″) were performed to study the self-settingprocess and gel point. The gel points are given as the time taken forthe liquid (G″>G′) to turn into a solid (G′>G″). They were determinedaccording to a derived percolation theory (Fatimi et al., ActaBiomater., 5: 3423-3432, 2009). Compressive modulus of scaffold wasmeasured using a TA HD-Plus (Stable Micro Systems). Six specimens weretested after three weeks of reticulation and the compressive modulus wascalculated on the basis of strain change from 0 to 5%. Shear strainmeasurements were performed with a Haake mars. Frequencies were appliedat a fixed total shear stress (1 Pa) and 0.21N. Oscillation tests wereperformed to measure G′ and G″ after 3 weeks of gelation. Nine specimenswere tested.

Cytocompatibility of Si-HPMC Hydrogel

Cellular Viability in 3D Culture

For 3D culture, MSC viability was quantitatively assessed by Live & Deadassays (Kit, Invitrogen, France) along with confocal image analysis.Briefly, MSCs were dispersed into the hydrogels within the 5 minutesfollowing their preparation at a final concentration of 1.10⁶ cells/mlof hydrogel. 250 μl of mixture was molded into ultra-low attachment24-well plates and incubated at 37° C. for 1 h to allow tie hydrogels tocrosslink. Afterwards, 500 μl of culture medium was added per well andthe samples were incubated for 24 h, 48 h and 7 days before Live & Deadassays were performed. In each well, the culture medium was replaced by200 μl of a solution containing 2.5 ml of culture medium supplementedwith 0.25 μl of calcein-AM (5 mM) and 5 μL of ethidium homodimer-1(EthD-1; 2 mM).

After 5 to 10 minutes, the dye mixture was removed and the hydrogelswere intensively rinsed with some phosphate buffered saline, beforebeing observed on a confocal laser-scanning microscope Nikon A1R (NikonFrance) equipped with an argon laser (488 nm) and a laser diode (561nm).

Images were recorded in 512×512 pixels with an objective CFI Plan FluorELWD 40X×40 LD NA:0.6. Resonant mode was used in bidirectionnel scanningwith average 16. For each sample, 6 random positions (x,y,z) were chosenwithin the hydrogel, and a stack of 100 planes were taken from these 6positions along the z axis with 10 μm step size.

Images obtained per sample were analyzed and the percentages of livingcells (in green) and dead cells (in red) were determined by using ImageJ(NIH) version 1.43u for Windows with the plugin “colour deconvolution”.Each condition was tested in triplicate, and each experiment wasrepeated three times.

Secretion of VEGF of MSCs in 3D Culture

Secretion of vascular endothelium growth factor (VEGF) in supernatantsfrom MSCs was quantified by specific enzyme-linked immunosorbent assay(ELISA) using a VEGF ELISA kit according to the manufacturer's protocol(R&D Systems, Minneapolis, Minn.). Briefly, MSCs were cultivated in 3dimensional into Si-HMPC hydrogel (10⁶ cells/ml of Si-HPMC hydrogel) andcell culture supernatant samples were collected from wells after 1, 2and 7 days of culture. MSCs cultivated in 2 dimensions without hydrogelwere used as control. Each condition was tested in triplicate, and eachexperiment was repeated three times.

Induction of MI in Rats and Implantation

Animal studies were performed in accordance with the regional EthicalCommittee CREEA (Comités régionaux d'éthique en matière expérimentationanimale). Female Lewis congenic rats (180-190 g) (Janvier France,) wereanesthetized with a mix of isoflurane/oxygen inhalation (3%/97%),incubated and ventilated (Harvard Rodent Ventilator, Harvard Apparatus).A left lateral thoracotomy in the fourth intercostals space wasperformed to expose the anterior surface of the heart. The proximal leftascending coronary artery was identified and ligatured with a 6.0polypropylene snare (Ethicon). The infarcted area was identified by thesurface scar and wall motion akinesis. Immediately after coronary arteryligation, a total of 150 μl of Si-HPMC hydrogel alone (hydrogel), MSCsalone (3.10⁶ cells) or in combination with the Si-HPMC hydrogel(MSC+hydrogel), or PBS (used as control), were delivered into themyocardium with a 26-gauge needle into 3 sites along the infarcted area.Sham-operated animals were subjected to the same surgical procedurewithout coronary artery ligation and injection. In all experiments, weat least 10 rats were used in each group.

Echocardioqraphic Measurements

Echocardiographic measurements were obtained at 1 day before MI(baseline), and 1 day and 7, 28 and 56 days after MI. Echocardiographicassessments were performed in-anesthetized rats (2% isofluraneinhalation) using a General Electric Vivid 7VR (GE Medical System;Milwaukee, Wis., http://www.gehealthcare.com) equipped with a 13-MHztransducer. Cardiac dimensions: Left ventricular end-diastolic diameter(LVEDD), end-systolic diameter (LVESD), and fraction shortening (LVFS)were recorded from M-mode images using averaged measurements from threeto five consecutive cardiac cycles according to the American Society ofEchocardiography. Left ventricular end-diastolic and end-systolicvolumes (LVEDV and LVESV, respectively) were calculated frombidimensional long-axis parasternal views taken through the infarctedarea by means of the single-plane area-length method (V=(8×A²)/(3×π×L)).LV ejection fraction (LVEF) was calculated as follows:LVEF=((LVEDV−LVESV)/LVEDV)×100. All measurements were averaged on threeconsecutive cardiac cycles and analyzed by a single observer who wasblinded to the treatment status of the animals.

Histopathology

Rat hearts were harvested, washed in PBS (pH 7.4) and fixed in 10%formalin for histology. Hearts were embedded in paraffin and 6 μmsections were cut from the apex to the level just below ligation. Threeevenly spaced sections were stained with Masson trichrome and observedwith a Nikon TE2000-E inverted microscope.

Circumferential extent of scar to total LV tissue (Kanashiro-Takeuchi RM et al., Proc. Natl. Acad. Sci. USA., 107: 2604-2609, 2010), relativescar thickness, and infarct expansion index (Ruvinov et al.,Biomaterials, 32: 565-578, 2011) were quantified using ImageJ (NIH)version 1.43u for Windows.

Average of epicardial and endocardial infarct ratios were calculated foreach section based on measurement of epicardial and endocardial infarctlengths and epicardial and endocardial LV circumference. For each heart,infarct size was calculated as the average of the value obtained for the3 analyzed sections. Relative scar thickness was calculated as averagescar thickness divided by average wall thickness, averaged from 3measurements of scar and septum thickness, respectively.average, Infarctexpansion index was calculated as follows: [LV cavity area/whole LVarea]/relative scar thickness. Percentage area of fibrosis in the remoteleft ventricle was quantified using an in-house image analysis programbase on the following formula: % fibrosis=fibrotic area/(fibroticarea+healthy area).

Statistical Analysis

All values are shown as mean±SEM. Comparative studies of means wereperformed by using one-way ANOVA followed by post-hoc test whenappropriate (Fisher's projected least significant difference) withp<0.05 as threshold for statistical significance. Echocardiographicparameters during 8-week follow-up were compared within groups andbetween groups using one-way ANOVA for repeated measurements followed bypost hoc tests, respectively. For a given parameter, p<0.05 wasconsidered significant. All tests were carried out using SigmaStat forWindows 3.5.

Results

Rheological Characteristics of Si-HPMC Hydrogel

Rheological properties of Si-HPMC solution mixed with acid buffer (1/1)were measured. The compressive modulus at 5% stress and the storagemodulus (G′) and loss modulus (G″) of Si-HPMC were performed after threeweeks of reticulation. The final product (Si-HPMC) consisted of areticulated hydrogel with a pH value of 7.4 after 27.2±3.4 min. Dynamicrheological measurements were performed to characterize this hydrogelincluding shear strain measurements to evaluate the storage modulus(G′), which characterizes the hard component, and the loss modulus (G″),which characterizes the liquid component. Compressive modulus, whichreflects the stiffness of the material in compressive experiment, was328.6±97.0 Pa. After three weeks of reticulation and a finishedself-setting process, a value of 343.2±106.5 Pa for the G′ and a valueof 44.5±15.4 Pa for the G″ were observed.

MSC Viability and Activity in Three Dimensional Culture within Si-HPMCHydrogel

To evaluate whether Si-HPMC hydrogel was cytotoxic, MSC viability wasquantified in 3D culture in Si-HPMC by conventional fluorescentmicroscopy (data not shown). MSC viability was maintained during thewhole culture period, from day 1 to day 7 (85.1±3.9% at day 1; 80.0±3.0%at day 2 and 74.3±3.9% at day 7; p=0.10 one-way ANOVA between groups)(FIG. 4).

To assess whether VEGF secretion was maintained in MSC 3D-culturedwithin Si-HPMC hydrogel for 7 days, VEGF concentrations were measured(ELISA) in supernatants at different time-points. Whereas VEGFconcentrations in the control supernatants (MSCs cultured withouthydrogel) were much higher (FIG. 5.A), VEGF concentration insupernatants from 3D-cultured MSCs within hydrogel increased overtimefrom 29.5±1.7 pg·ml⁻¹ at day 1 to 91.0±5.1 pg·ml−1 at day 2 to 181.2±6.4pg·ml⁻¹ at day 7; p<0.001 for all comparisons) (FIG. 5.B).

Comparative Effects of Hydrogel, MSC, and MSC+Hydrogel on CardiacFunction and LV Remodeling

MI was induced in 62 rats by ligation of the left anterior descendingcoronary artery. After MI induction, rats were randomised into 4treatment groups to receive intramyocardial injections of (1) PBS ascontrol, (2) Si-HPMC hydrogel alone (hydrogel), (3) MSCs alone (MSC) and(4) Si-HPMC hydrogel loaded with MSCs (MSC+hydrogel). Overall mortalityat 24 hours after surgery was 30.7±7.7% (19/62 rats) with no significantdifferences between treatment groups (see below Table 1A).Echocardiography was performed 1 day after coronary ligation, to selectrats with a significant myocardial infarction so as to maximize possibletreatment effects (defined as animals with LVEF≤70%; table 1B). Thenumber of selected rats was not significantly different betweentreatment groups (see below Table 1B). Importantly, parameters of leftventricular (LV) dimensions and function measured at day 1 were notdifferent between the 4 treatment groups in the animals entering theechocardiography follow-up study (See below Table 2).

TABLE 1A Animals Living number at animals baseline at day 1 PBS 11 10hydrogel 14 11 MSC 15 9 MSC + hydrogel 22 13 Total 62 43

TABLE 1B Animals Animals number number with with LVEF > 70% LVEF < 70%at day 1 at day 1 PBS 4 6 hydrogel 4 7 MSC 1 8 MSC + hydrogel 4 9 Total13 30

TABLE 2 PBS hydrogel MSCs MSC + hydrogel Parameter (n = 6) (n = 7) (n =8) (n = 9) LVEDD (mm) Bsl  5.4 ± 0.2  5.2 ± 0.2 5.6 ± 0.1 5.6 ± 0.2 d1 5.9 ± 0.1  5.8 ± 0.3 6.2 ± 0.2 6.0 ± 0.1 d7  6.6 ± 0.1  6.1 ± 0.3 6.3 ±0.1 6.1 ± 0.2 d28   7.2 ± 0.2^(¥)   6.9 ± 0.3^(¥) 7.0 ± 0.3 6.6 ± 0.3d56   7.4 ± 0.3^(¥)   7.3 ± 0.5^(¥) 7.0 ± 0.4 6.8 ± 0.2 LVESD (mm) Bsl 2.4 ± 0.1  2.6 ± 0.2 2.8 ± 0.1 3.0 ± 0.2 d1  4.0 ± 0.1  4.1 ± 0.3 4.3 ±0.1 4.3 ± 0.2 d7   5.0 ± 0.1^(¥)   4.0 ± 0.3 *  4.3 ± 0.2 *  3.9 ± 0.2 *d28   5.7 ± 0.3^(¥)   5.0 ± 0.3 *  5.2 ± 0.4 *  4.3 ± 0.3 * d56   6.0 ±0.3^(¥)   5.5 ± 0.5 *  4.9 ± 0.3 *    4.8 ± 0.1 * ^($) FS (%) Bsl 56.6 ±1.7 49.4 ± 2.0 49.5 ± 1.0  47.1 ± 2.2  d1 29.0 ± 2.4 29.9 ± 2.8 30.4 ±1.8  27.9 ± 1.9  d7 24.1 ± 0.9  34.1 ± 2.0 * 31.2 ± 2.5    36.9 ±1.7^(¥) * ⁺ d28 20.2 ± 2.3  28.0 ± 1.2 *  26.7 ± 3.3 *    34.4 ±1.9^(¥) * ⁺ ^($) d56 19.6 ± 1.5  25.6 ± 2.9 *  30.8 ± 2.4 *  29.4 ±1.5 * ^($) EF (%) Bsl 87.4 ± 1.5 86.0 ± 1.2 86.8 ± 1.9  88.2 ± 1.5  d161.3 ± 4.0 64.6 ± 2.6 64.6 ± 1.8  61.2 ± 2.9  d7 55.7 ± 2.4 68.0 ± 2.363.5 ± 3.2     76.0 ± 1.6^(¥) * + d28 49.0 ± 2.5  71.7 ± 2.6 *  72.4 ±1.5 *  76.4 ± 1.5^(¥) * d56 47.4 ± 2.4 56.9 ± 4.6  65.4 ± 3.3 *   68.5 ±2.0^(¥) ^($)

As expected in the PBS group, MI led to a time-dependent increase in LVchamber dimensions (LVEDD: 5.9±0.1 mm at day 1 vs 7.4±0.3 mm at day 56;p<0.05. LVESD: 4.0±0.1 mm at day 1 vs 6.0±0.3 mm at day 56; p<0.05.)(FIGS. 6.A and 6.B) and reduction in EF (61.3±1.5% at day 1 vs 47.4±2.4%at day 56; p<0.05) (FIG. 6.D) and fraction shortening (FS) (29.0±2.4% atday 1 vs 19.6±1.5% at day 56; p<0.001) (FIG. 6.C). As compared to PBSgroup injections of hydrogel, MSC or MSC+hydrogel significantlyattenuated the MI-induced increase of LV end-systolic diameter (LVESD)(FIG. 6.B) and reduction of FS (FIG. 6.C) and EF (FIG. 6.D).Interestingly, significant differences were observed between these 3groups:

(1) In the hydrogel groups, LVEF was significantly increased at 28 daysafter injection as compared to PBS group (71.7±2.6% vs 49.0±2.5%;p<0.001) but not at day 7 and day 56. In addition, the LVESD was reducedduring the whole study as compared to PBS group but not the LVEDD. TheLVESD, the LVFS and the LVEF were not significantly altered during thewhole study as compared to day 1 but the LVEDD was increased at day 28and 56 as compared to day 1 (7.3±0.5 mm at day 56 vs 5.8±0.3 mm at day1; p<0.001).

(2) In the MSC group, LVEF was significantly increased at 28 and 56 daysafter injection as compared to PBS group (at day 56: 65.4±3.3% vs47.4±2.4%; p<0.001), but not at day 7. In addition, the LVESD wasreduced during the whole study as compared to PBS group but not theLVEDD. The LVESD, the LVEDD, the LVFS and the LVEF were notsignificantly altered during the whole study as compared to day 1.

(3) In the MSC+hydrogel group LVEF was significantly increased at day 7up to day 56 after injection as compared to PBS group (at day 7:76.0±1.6% vs 55.7±2.4%; p<0.001). In addition, the LVESD was reducedduring the whole study as compared to PBS group but not the LVEDD. TheLVFS and the LVEF were significantly increased compared to day 1(61.2±2.9%) at 28 days (76.4±1.5%; p<0.001) then maintained at 56 days(68.5±2.0%; p=0.05).

Interestingly the LVEF was higher at day 7 as compared to LVEF in MSCgroup (76.0±1.6% vs 63.5±3.2%; p<0.05) and at day 56 in compared tohydrogel group (68.5±2.0% vs 56.9±4.6%; p<0.05). Similar results wereobserved for LVFS (FIG. 6C).

Comparative Effects of Hydrogel, MSC or MSC+Hydrogel on InfarctExpansion and Ventricular Fibrosis.

Morphometric analysis of heart sections was performed to analyse LVremodeling. For all animals, Infarct area was located in the anteriorregion of the left ventricle (FIG. 7A).

The MI size had been reduce as compared to the PBS group (53.8±2.5%) inthe hydrogel group (43.0±4.2%; p<0.05), in the MSC group (35.2±1.5%;p<0.001) and in the MSC+hydrogel group (28.2±1.2%; p<0.001) (FIG. 7.B).In addition the MI size was reduced in MSC and MSC+hydrogel groups ascompared to the hydrogel group.

The percentage of ventricular fibrosis (FIG. 7.C) was significantlyreduced as compared to the PBS group (27.8±1.6%) in the hydrogel group(19.0±2.5%; p<0.05), MSC group (7.9±0.6%; p<0.001) and MSC+hydrogelgroup (6.7±0.6%; p<0.001).

Relative scar thickness (FIG. 8.B) was significantly increased ascompared to PBS group (0.36±0.05) in hydrogel group (0.53±0.04; p<0.05),MSC group (0.59±0.04; p<0.05), and MSC+hydrogel group (0.63±0.04;p<0.001).

Infarct expansion index (FIG. 8.C) was significantly decreased ascompared to PBS group (1.73±0.24) in hydrogel group (0.97±0.09;p<0.001), MSC group (0.81±0.04, p<0.001), and MSC+hydrogel (0.66±0.04;p<0.001).

Interestingly, chondroid metaplasia of the endocardium was observed in83% rats in PBS group (FIG. 8.A) whereas this feature was visible in 67%rats in the hydrogel group, 60% rats in the MSC group, and only in 14%in rats in hydrogel+MSC groups.

Taken together, these results show that (1) hydrogel neither altered MSCviability nor activity and (2) injection of Si-HPMC hydrogel load withMSCs in the heart directly after MI leads to cardiac function and LVremodeling preservation as compared to hydrogel or MSCs alone.

The invention claimed is:
 1. A kit for obtaining an aqueous compositionthat is usable in a method of treating heart failure, said kitcomprising i) mesenchymal stem cells which are able to differentiateinto cardiomyocytes, and ii) a hydrogel solution which comprises asilylated biomolecule capable of forming a pH dependentself-reticulating hydrogel, wherein the hydrogel solution comprisesagents which induce cardiomyocyte differentiation of the mesenchymalstem cells.
 2. The kit according to claim 1, further comprisinginstructions for the use of said kit in preparing a compositioncomprising: i) the mesenchymal stem cells which are able todifferentiate into cardiomyocytes; and ii) the hydrogel solutioncomprising a silylated biomolecule; said composition being suitable forinjection into myocardium.
 3. The kit according to claim 1, wherein thesilylated biomolecule is selected from the group consisting of asilylated polysaccharide, a silylated peptide a silylated protein, andan association of two different biomolecules selected from a silylatedpolysaccharide, a silylated peptide, and a silylated protein.
 4. The kitaccording to claim 3, wherein the silylated polysaccharide is selectedfrom the group consisting of: silylated cellulose, silylatedhydroxypropylmethylcellulose (HPMC), silylated hydroxyethylcellulose(HEC), silylated carboxymethylcellulose (CMC), silylated pectin,silylated chitosan and silylated hyaluronic acid.
 5. The kit accordingto claim 1, wherein the hydrogel solution of (ii) has undergone threeweeks of reticulation and has the following rheological characteristicsat a pH value of 7.4: a compressive modulus at 5% stress from 220 to 430Pa; a storage modulus G′ from 235 to 450 Pa; a loss modulus G″ from 29to 60 Pa; a gel point from 23.8 to 30.6 minutes.
 6. A kit for thethree-dimensional culture of mesenchymal stem cells which are able todifferentiate into cardiomyocytes, said kit comprising i) mesenchymalstem cells which are able to differentiate into cardiomyocytes, and ii)a hydrogel solution which comprises a silylated biomolecule capable offorming a pH dependent self-reticulating hydrogel, wherein the hydrogelsolution comprises agents which induce cardiomyocyte differentiation ofthe mesenchymal stem cells.
 7. The kit according to claim 6, wherein thesilylated biomolecule is selected from the group consisting of asilylated polysaccharide, a silylated peptide a silylated protein, andan association of two different biomolecules selected from a silylatedpolysaccharide, a silylated peptide, and a silylated protein.
 8. The kitaccording to claim 7, wherein the silylated polysaccharide is selectedfrom the group consisting of: silylated cellulose, silylatedhydroxypropylmethylcellulose (HPMC), silylated hydroxyethylcellulose(HEC), silylated carboxymethylcellulose (CMC), silylated pectin,silylated chitosan and silylated hyaluronic acid.
 9. The kit accordingto claim 6, wherein the hydrogel solution (ii) has undergone three weeksof reticulation and has the following rheological characteristics at apH value of 7.4: a compressive modulus at 5% stress from 220 to 15 000Pa; a storage modulus G′ from 235 to 10 000 Pa; a loss modulus G″ from29 to 1000 Pa; a gel point from 5 to 45 minutes.